In vitro evaluation suggests fenfluramine and norfenfluramine are unlikely to act as perpetrators of drug interactions

Abstract Studies support the safety and efficacy of fenfluramine (FFA) as an antiseizure medication (ASM) in Dravet syndrome, Lennox‐Gastaut syndrome, or CDKL5 deficiency disorder, all pharmacoresistant developmental and epileptic encephalopathies. However, drug–drug interactions with FFA in multi‐ASM regimens have not been fully investigated. We characterized the perpetrator potential of FFA and its active metabolite, norfenfluramine (nFFA), in vitro by assessing cytochrome P450 (CYP450) inhibition in human liver microsomes, CYP450 induction in cultured human hepatocytes, and drug transporter inhibition potential in permeability or cellular uptake assays. Mean plasma unbound fraction was ~50% for both FFA and nFFA, with no apparent concentration dependence. FFA and nFFA were direct in vitro inhibitors of CYP2D6 (IC50, 4.7 and 16 µM, respectively) but did not substantially inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5. No time‐ or metabolism‐dependent CYP450 inhibition occurred. FFA and nFFA did not induce CYP1A2; both induced CYP2B6 (up to 2.8‐fold and up to 2.0‐fold, respectively) and CYP3A4 (1.9‐ to 3.0‐fold and 3.6‐ to 4.8‐fold, respectively). Mechanistic static pharmacokinetic models predicted that neither CYP450 inhibition nor induction was likely to be clinically relevant at doses typically used for seizure reduction (ratio of area under curve [AUCR] for inhibition <1.25; AUCR for induction >0.8). Transporters OCT2 and MATE1 were inhibited by FFA (IC50, 19.8 and 9.0 μM) and nFFA (IC50, 5.2 and 4.6 μM) at concentrations higher than clinically achievable; remaining transporters were not inhibited. Results suggest that FFA and nFFA are unlikely drug–drug interaction perpetrators at clinically relevant doses of FFA (0.2−0.7 mg/kg/day).


| INTRODUC TI ON
most patients are prescribed three to four concurrent ASMs. [4][5][6][7] During the FFA development program, the potential for drugdrug interactions (DDIs) when FFA is added to existing ASM regimens was evaluated in vivo. Initial treatment for patients with LGS is usually valproate, lamotrigine, and/or topiramate, followed by adjunctive felbamate, clobazam, levetiracetam, and cannabidiol. 5,8 Patients with DS typically are first prescribed valproate and clobazam, with refractory seizures treated by adding stiripentol. 9 Adjunctive cannabidiol and FFA are more recently developed treatment options 2,3,5 ; clonazepam, levetiracetam, and zonisamide, with ethosuximide for atypical absence seizures, 9 are additional options.

CDD does not currently have a targeted therapy, although recent
open-label trials support clinical efficacy of both cannabidiol and FFA in reducing median convulsive seizure frequency. 10,11 FFA undergoes de-alkylation to norfenfluramine (nFFA) 12,13 in the liver by the cytochrome P450 (CYP450) mixed-function oxidase system. Therefore, both FFA and nFFA were evaluated for their drug interaction potential. CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 catalyze most drug biotransformation reactions. These enzymes are commonly implicated in DDIs. 14,15 The pharmacokinetics of ASMs used to treat LGS and/ or DS, such as clobazam and cannabidiol, may have victim potential when used in combination with moderate or strong CYP450 inhibitors and may require dose adjustments due to DDIs. 16,17 Polypharmacy is typically used to manage the multiplicity of seizures characteristic of developmental and epileptic encephalopathies. FFA will be used in combination regimens, necessitating a thorough characterization of potential for DDIs. The FDA and EMA have developed comprehensive "Guidance for Industry" documents which outline the most current state of the science for predicting DDIs. These documents use "a systematic, risk-based approach to assessing DDI potential of investigational drugs and making recommendations to mitigate DDIs." 15 The recommended assays provide a robust, rigorous, systematic investigation into both victim and perpetrator potential of an investigational drug. The outcome of these experiments is aimed to provide regulatory support for the investigational product and inform clinical practice when prescribing FFA in combination ASM regimens.
Perpetrator (or precipitant) ASMs may affect the clearance, efficacy, and/or toxicity of victim (or object) ASMs in combination therapy, especially if the perpetrator induces or inhibits an enzyme responsible for a single-elimination pathway. [18][19][20] Perpetrator ASMs can also inhibit drug transporter proteins widely distributed throughout the body, thereby modulating drug pharmacokinetics and drug action via modifications in absorption, distribution, tissue-specific drug targeting, and elimination of the victim drug. 21 US FDA guidance for industry recommends that sponsors conduct a comprehensive panel of in vitro metabolism-and transportermediated DDI studies as part of their preclinical development programs. 15 In this study, we performed in vitro DDI studies in accordance with FDA guidance to identify the CYP450 enzymes and transporter proteins that could be altered and contribute to the perpetrator potential of FFA and its major metabolite, nFFA, in the context of multi-ASM dosing regimens. 15

| Direct CYP450 inhibition
Inhibition of CYP450s by FFA and nFFA was assessed according to methods described previously. 19

| Metabolism-dependent CYP450 inhibition
To examine metabolism-dependent inhibition of the CYP450 enzymes, FFA and nFFA were preincubated in duplicate at 37 ± 1°C with human liver microsomes as described above for ~30 min in the presence of an NADPH-generating system, which allowed for the generation of potential intermediates that could irreversibly inhibit human CYP450 enzymes. For comparison, additional duplicate samples containing FFA or nFFA were preincubated for 30 min in the absence of NADPH. These preincubations allowed assessment of the NADPH-dependence of any time-dependent CYP450 inhibition.
Following the 30-min preincubation, the NADPH-generating system and/or marker substrate was added, and incubations were continued as described previously to measure residual CYP450 enzyme activity. Incubations that contained FFA and nFFA but were not preincubated served as negative controls for time-dependent CYP450 inhibition.

| Red blood cell/plasma partition
To determine the extent of red blood cell accumulation of FFA and nFFA, the red blood cell/plasma partition of both drugs was assessed in human blood obtained from BioIVT (Westbury, NY where:

| CYP450 induction
Measurement of CYP450 induction in human hepatocyte cultures was performed as previously described. 27 Briefly, cultures were obtained from three lots of cryopreserved hepatocytes (Sekisui XenoTech, Kansas City, KS) and were treated once daily for 3 con- Cat. # 11644793001) and based on daily microscopic evaluation.  Co. Ltd., Tokyo, Japan) were used to assess FFA and nFFA inhibitory effects as described. 29 The test compound (FFA or nFFA) or the positive control substrate (digoxin and prazosin for BCRP and P-gp transporters, respectively) was added to the donor chamber.  Table 1).

| Statistical analysis and data analysis
Summary data are expressed as percent of control and mean ± standard deviation (SD) or standard error of the mean (SEM), as appropriate. Individual values from inhibition studies were processed with the laboratory information management system (LIMS) Galileo

| CYP450 inhibition
The inhibitory effects of FFA and nFFA on CYP450 enzymes in terms of IC 50 Figure 1C). nFFA inhibited up to 2.8% of CYP3A4/5 testosterone 6β hydroxylase activity. Under experimental conditions to measure time-or metabolism-dependent inhibition, FFA and nFFA did not have any additional inhibitory effects on CYP450 enzyme activity compared to direct inhibition ( Figure 1B, D for CYP2D6).
FFA and nFFA potential to inhibit CYP450 enzymes in the clinic was evaluated initially with the basic model and then with the static mechanistic model. 15  was also unlikely to be necessary. The FFA potential to inhibit intestinal CYP3A4/5 was evaluated with the basic model. The calculated R 1,gut value was <1.8 and was lower than the threshold value of 11, indicating that clinically relevant inhibition of CYP3A4/5 in the gut by FFA was unlikely.

| CYP induction
At the time of isolation for cryopreservation, the viability of hepatocyte preparations used for induction assays was between 81.7% and 91% (Supplemental Table 3 (Table 2). These values, according to the fold-change method, indicate lack of potential to induce the enzyme in vivo. 15 No further estimates were con-     Figure 2).
To further investigate the potential for FFA and nFFA to induce CYP2B6 and CYP3A4 clinically, basic kinetic and static mechanistic models were applied. E max and EC 50 parameters for effects of FFA and nFFA on the fold-increase in CYP2B6 and CYP3A4 mRNA were estimated from sigmoidal three-parameter equations. The parameters were applied to calculate R 3 and AUCR values for the basic kinetic and static mechanistic models, respectively (Table 2; Supplemental Table 2). 15 The R 3 value predicted CYP2B6 induction by FFA in two of three hepatocyte cultures but did not predict CYP2B6 induction by nFFA (

| Transporter inhibition
The IC 50 values characterizing inhibition of P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2-K transporters by FFA and nFFA, as well as the FDA recommended criteria for evaluation of drug transporter inhibition, namely, the formulae for R values and their cutoffs, are presented in Table 3. 15   Our in vitro results suggest that FFA is unlikely to affect drug distribution and/or elimination by inhibiting the major drug transporters. 46 The R values were below the FDA-specified threshold for transporter inhibition of OATP1B1 and OATP1B3 hepatic uptake transporters or OAT1, OAT3, OCT, or MATE2-K renal transporters (Table 3). 15 FFA and nFFA inhibition of renal clearance transporters OCT2 and MATE1 47 was greater than that observed for the other transporters investigated (Table 3). However, the observed inhibi-  FFA is a racemic mixture of two enantiomers. Although prior publications have evaluated the enantiomers separately, the clinically used FFA is administered as a racemate and therefore the experimental studies were conducted with the racemic form.

| DISCUSS ION
This study has some limitations. It should be noted that pharmacogenetic variants cause affected individuals to be classified as poor, extensive, or ultra-metabolizers of CYP2D6 substrates, as well as CYP2C9, CYP2C19, and CYP3A4 substrates, 52 although data suggest that polymorphism of any individual CYP450 is unlikely to affect FFA pharmacokinetics (Zogenix, data on file). It is unknown how interindividual differences among pharmacogenetic variants of CYP450s and drug transporters could affect local and systemic drug concentrations of FFA.
In conclusion, this study provides a comprehensive examination of the impact of FFA and nFFA on clinically relevant CYP450s and transporter proteins. The in vitro DDI data suggest limited potential for FFA to have significant perpetrator activity in multidrug ASM regimens.

ACK N OWLED G EM ENTS
The authors thank scientists from Enzyme Incubations, Analytical Sciences and Data Processing groups at Sekisui XenoTech for their technical expertise in execution of this study.

E TH I C A L S TATEM ENT
This study is exempt from ethics approval.

DATA AVA I L A B I L I T Y S TAT E M E N T
Zogenix is in the process of establishing a data sharing policy. Written requests for data by legitimate investigators/researchers/clinicians may be submitted to Zogenix, Inc. These requests will be considered on a case-by-case basis and reviewed for appropriateness.